The human cancers, which often originate from a single malignantly transformed cell, could be caused by exposure to environmental carcinogens. Since the dose of carcinogens in the environment is substantially low, it is assumed that cellular events leading to transformation cannot readily be detected by currently existing carcinogenicity (in vivo) or genotoxicity (in vitro) tests. Thus, it is still not known well how frequently such transformations are induced by environmental carcinogens. As a compromise, high and lethal doses of test substances have been used in laboratory tests and results obtained with high doses are used to extrapolate the likely effects of lower doses and assess carcinogenic potential. Conceptually, such extrapolation could create complications in the interpretation of results. Firstly, cytotoxicity caused by lethal or high doses could prevent the detection of carcinogenic events, as malignantly transformed cells, which are supposed to be the focus of studies, are also eliminated by cytotoxicity. Secondly, there is no solid evidence that the critical alterations that occur in a small number of cells following exposure to low doses can indeed be extrapolated from responses of cells induced by lethal doses, which could alter the majority of cells. If responses of cells induced by low doses are distinct from that of high doses, extrapolation could lead to inaccurate conclusions. At this moment, there is almost no accurate way to know whether conclusions obtained through extrapolation are correct, as information regarding the responses of cells to low and environmental doses of carcinogens is not available.
One of well-known in vitro tests, the Ames test, which uses histidine deficient salmonella mutants and evaluates genotoxicity by counting the number of revertants produced as a result of gene mutations shows a failure rate of about 55%. Various mammalian cell-based assays, including the sister chromatid exchange test, the mouse lymphoma tk gene mutation assay and the chromosomal aberration test, show good positive rates for known rodent carcinogens (about 70-90%), but also show high positive rates for non-carcinogens (false positive rates; over 70-80%), thereby making the accurate assessment of the carcinogenicity of substances difficult.
In vivo test, i.e. carcinogenicity tests with animals, mainly with rats and mice, also suffers inaccuracy. The life span of rodents is less than 2-3 years. Thus, in order to produce a tumor, which growth in humans often takes over 20 years, within the life time of experimental rodents, high doses or close to lethal doses of substances are often administrated despite concerns that such high doses do not represent the situation in humans that are exposed to substantially lower doses of carcinogens. In fact, it has been known that false positive or negative results are likely produced by carcinogenicity tests with experimental animals. Thus, human carcinogens are generally difficult to identify by existing laboratory-based investigations. All of these problems could be related to the lack of information regarding cell responses induced by environmental doses of carcinogens.
There is thus a need for a non-extrapolation method that allows the study of abnormal and rare cellular events induced by environmental doses of carcinogens. Such events could be identified and detected by video recording of live cells (live-cell imaging), tracking of individual cells, creation of cell lineage maps, establishment of cell lineage database and quantitative identification of abnormal and rare cellular events. The majority of currently available live-cell imaging analyses are based on visualization of cells through excitation of fluorophores, e.g. fluorescent proteins (green, red, yellow, etc.), fluorescent dyes, quantum dots, etc. However, excitation of fluorophores is known to be a cause of phototoxicity, hindering the detection of abnormal and rare cellular events.
There is thus a need for a fluorescence-free live-cell imaging technique useful in the determination of quality of mammalian cells and for the study of carcinogenicity of compounds, especially response to low/veritable and environmental doses of carcinogens.
The present invention addresses these needs, as it relates to methods and biomarkers useful in the assessment of compounds, screening of compounds, evaluation and/or control of the quality of cells, etc.
Additional features of the invention will be apparent from review of the disclosure, figures, and description of the invention.